Formation Mechanism of Flattened Top HFBI Domical Droplets - The

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Formation Mechanism of Flattened Top HFBI Domical Droplets Ryota Yamasaki†,‡,§ and Tetsuya Haruyama*,†,‡,§ †

Division of Functional Interface Engineering, Department of Biological Functions Engineering, and ‡Research Center for Eco-fitting Technology, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Fukuoka 808-0196, Japan § Advanced Catalytic Transformation Program for Carbon Utilization (ACT-C), Japan Science and Technology Agency (JST), Tokyo 102-0076, Japan ABSTRACT: A water droplet assumes a spherical shape because of its own surface tension. However, water droplets containing dissolved hydrophobin (HFBI) have flat surfaces. In our previous study, the mechanism of this unique phenomenon was revealed. HFBI forms a self-organized membrane that has a densely packed and honeycomb-like structure. Furthermore, the buckling strength of the membrane is higher than the surface tension of the HFBI droplet. Therefore, an HFBI domical droplet has a flat surface. However, it was not clear why only the top of the domical droplet was flattened while other areas such as the side face were not. In this study, we observed HFBI domical droplets to investigate this phenomenon. The flat top area (self-organized HFBI membrane) remained parallel to the ground even if the substrate was tilted. Therefore, buoyancy was thought to be a factor affecting the HFBI membrane. In addition, the side face of the HFBI domical droplet was analyzed by atomic force microscopy and electrochemical impedance spectroscopy, and it was found that the sides of the HFBI droplet were not composed of densely packed HFBI membranes.

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INTRODUCTION A water droplet assumes a spherical shape because of its surface tension, which is defined as the surface free energy per unit area. Observation of the air/water interface at a molecular scale revealed that water molecules in the bulk have a low free energy because of the omnidirectional intermolecular force exerted by the other molecules. Meanwhile, water molecules on the surface are subjected to interactions from the interior of the water phase, but are minimally affected by the molecules in the air phase. Therefore, water molecules have a large surface free energy on the surface as compared to the interior, and the surface area tends to be as small as possible. Consequently, the water droplets take the shape of a sphere that has the smallest surface area to volume ratio.1 However, water droplets containing dissolved HFBI display a unique phenomenon. Instead of assuming a spherical shape, the tops of the HFBIdissolved water droplets have flat surfaces.2,3 HFBI belongs to the class of hydrophobin proteins, which are small surface-active proteins having various roles in fungal growth and development. Hydrophobins lower the surface tension of water to assist the growth of the fungal hyphae through the air/water interface.4,5 Hydrophobins comprise about 100 amino acids and include four disulfide bonds between eight conserved cysteine residues.6,7 Therefore, they have a remarkably stable structure and can endure temperatures close to the boiling point of water.8 Hydrophobins are segregated into class I and class II according to the hydropathy patterns of their sequence and the solubility of the assembled layers.9,10 Class I hydrophobins appear to be more resistant to solvents and detergents as compared to class II hydrophobins.11 © 2016 American Chemical Society

Furthermore, class I hydrophobin layers tend to form a rodlet structure at interfaces, whereas class II hydrophobins do not.12,13 HFBI is a class II hydrophobin derived from Trichoderma reesei, and it is prone to self-assembly. HFBI is a relatively small protein with a molecular mass of about 7.5 kDa. As an amphiphilic protein, it forms a self-organized membrane at both air/water and water/solid interfaces. When HFBI forms a self-organized membrane at an air/water interface, the top of the water droplet is flattened.2,3 This unique phenomenon has been clarified in our previous paper. The self-organized HFBI membrane has a precise honeycomb-like structure with a buckling strength higher than the surface tension of the HFBIdissolved water droplet,3,14 as revealed by force curve measurements. Therefore, the top of the HFBI-dissolved water droplet becomes flat. However, the reason for the same is not yet understood. In this paper, we clarify the details of the driving force acting on the HFBI protein in the liquid and the processes occurring on the sides of the HFBI droplet. In this regard, we carefully observed the HFBI droplet on a tilted substrate, and measured the top and side air/water interfaces of the HFBI droplet by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS).

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EXPERIMENTAL SECTION HFBI-Dissolved Water Droplet. The HFBI protein from Trichoderma reesei was isolated and purified as previously Received: February 8, 2016 Revised: April 1, 2016 Published: April 2, 2016 3699

DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704

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The Journal of Physical Chemistry B reported.15 A 1 mM acetate buffer (pH 5.0) consisting of acetic acid and sodium acetate (purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan)) was used to dissolve the proteins. HFBI was dissolved in 1 mM acetate buffer at a 15 μM concentration. The HFBI solution was sonicated at 28 kHz for 1 min to disperse the protein in the solution. A domical water drop was prepared on a hydrophobic glass substrate (fluorine-doped tin oxide (FTO), purchased from Asahi Glass Co. (Tokyo, Japan)) using 10 μL of the HFBI solution. AFM Measurements. Surface structure analysis of the selforganized HFBI membrane was performed by AFM, a measurement technique giving subnanometer spatial resolution in the vertical and lateral directions.16 Since the tip-to-sample distance is regulated by detecting the tip-to-sample interaction forces, AFM can be used for investigations even on insulators. AFM has recently been used to analyze relatively soft samples such as polymers and biological samples17−19 and is therefore suitable for analysis of the self-organized HFBI membrane. The structure of the self-organized HFBI membrane has already been mentioned in a previous paper.13,20−23 In this study, AFM was used to investigate the sides of the HFBI domical droplet. First, the solution was sonicated at 28 kHz for 1 min to disperse the protein. Droplets with a volume of 10 μL were formed using a pipet. After 15 min, the HFBI formed a self-organized membrane at the air/water interface. Next, highly oriented pyrolytic graphite (HOPG) (10 mm × 10 mm × 1 mm, grade SPI-1, purchased from Structure Probe, Inc. (West Chester, PA)) was cleaved with Scotch tape to yield a clean surface. Finally, the substrate was brought in contact with the top and side faces. These substrates were rinsed in deionized and distilled water. The modified substrate was imaged in a liquid, and the measurements were performed in a buffer (100 mM phosphate buffer, pH 7.0), where 100 μL of the buffer was loaded onto the substrate. In the liquid measurements, a PPP-NCHAu probe (42 N/m, Nanoworld) was used for imaging. Imaging was done in the tapping mode. Electrochemical Measurements. EIS was performed using an electrochemical polarizer (Hokuto Denko Co., Japan) with a platinum wire as the counter electrode, a Ag/ AgCl wire as the reference electrode, and HOPG as the working electrode. The electrolyte was freshly prepared 0.1 M phosphate buffer (pH 7.0). All potentials were reported with respect to Ag/AgCl. EIS was performed on HOPG electrodes covered with HFBI in a 0.1 M phosphate buffer solution containing two types of redox molecules, i.e., [Fe(CN)6]3−/4−. Measurements were carried out at room temperature (25 °C). Impedance spectra in the range of 105−10−1 or 105−10−2 Hz were recorded with a 10 mV root-mean-square amplitude potential perturbation. Observation of the HFBI Domical Droplet. The HFBI domical droplet on FTO was observed by a CAM 200 (KSV Instruments Ltd., Finland) instrument. The domical drop was incubated at room temperature in a humidified atmosphere. The observation stage was shielded and maintained at saturated humidity, which limited evaporation of the domical drops.

Figure 1. (a) HFBI molecular model. HFBI is a globular molecule with a size of about 2 or 3 nm. (b) Photographs of the domical shape of an HFBI-dissolved water droplet (left) and a flattened droplet, in which an HFBI self-organized structure has developed at the surface (right). (c) AFM image of a self-organized HFBI membrane. The substrate was prepared to be transferred to the top of a domical droplet.

interface was transferred to the HOPG, and its surface structure was measured by AFM. The structure of the self-organized HFBI membrane has already been mentioned in a previous paper.1,3,20−23 In this study, it was also found that the selforganized HFBI membrane has a precise honeycomb-like structure (Figure 1c). A hexagonal structure can form either a planar surface or a tube, but not a sphere. Moreover, the buckling strength of the self-organized HFBI membrane was determined by force curve measurements.3 It was found that the self-organized HFBI membrane has a buckling strength higher than the innate surface tension. Hence, the top of the HFBI droplet has a flat surface. However, the reason for this flattening at the top surface remains to be understood. Generally, in the case of a hydrophobic substrate, a water droplet forms a high contact angle. Once the water evaporates, the water droplet shrinks but remains unchanged in shape because the hydrophobic surface is hard to get wet (Figure 2a). In the case of the hydrophilic substrate, the water droplet results in a low contact angle. Once the water evaporates, the water droplet again shrinks, but the width of the water/solid interface is unchanged because the hydrophilic surface is easy to get wet (Figure 2b). However, the HFBI droplet first forms a high contact angle on the hydrophobic substrate. Subsequently,

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RESULTS AND DISCUSSION Droplet Interface Phenomenon. In general, the water droplet forms a sphere; the HFBI-dissolved water droplet, however, forms a flattened top instead of a sphere (Figure 1b). In our previous study, this HFBI droplet interface phenomenon was revealed to be due to self-organization at the air/water interface. The self-organized HFBI membrane at the air/water 3700

DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704

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The Journal of Physical Chemistry B

Figure 2. Theoretical behavior of a water droplet on two types of substrates: hydrophobic (a) or hydrophilic (b). (c) Behavior of an HFBI domical droplet on a hydrophobic substrate.

the droplet/substrate (water/solid) interface becomes hydrophilic because of the adsorption of HFBI. The HFBI droplet does not change the width of the water/solid interface, as shown in Figure 2b. Therefore, only the top of the HFBI droplet can change in shape (Figure 2c). Analysis of the Side Face of a Self-Organized HFBI Droplet. The formation of a self-organized HFBI membrane on the top surface of the droplet was confirmed. However, the side face of the HFBI droplet had not been investigated. First, AFM and EIS measurements were carried out to analyze the side face of the droplet. The top and side of the self-organized HFBI droplet were transferred to the substrate. The transferred substrates were subjected to AFM and EIS (Figure 3). The transferred substrate from the top face of the droplet was confirmed to be a very precise self-organized HFBI membrane (Figure 3a). On the other hand, there was no self-organized HFBI membrane from the side of the droplet, but only a region sparsely populated by the proteins (Figure 3b). These substrates were investigated by EIS (Figure 3c). The EIS data are shown in the Nyquist plot (“Zim” and “Zre”). The diameters of these semicircle plots correspond to the charge transfer resistance: the larger the semicircle diameter, the higher the charge transfer resistance. As a result, the substrate transferred to the top face of the droplet experienced high charge transfer resistance because of the large number of protein molecules present on the substrate. However, the substrate transferred to the side face of the droplet experienced lower charge transfer resistance. From these results, it is clear that the top of the HFBI droplet has a densely packed selforganized HFBI membrane and that the side of the droplet has no such membrane. Behavior of an HFBI Droplet on a Tilted Substrate. The top of an HFBI-dissolved water droplet on a horizontal substrate was flattened (Figure 1b). In our previous study, it was revealed that the top of the HFBI droplet was flattened because the self-organized HFBI membrane has a strong and honeycomb-like structure. However, this does not clarify why the HFBI droplet is flattened from its apex. It is likely that

Figure 3. AFM and EIS measurements: (a) AFM image of the substrate transferred to the top face, (b) AFM image of the substrate transferred to the side face, (c) EIS measurement of the substrate transferred to both the top and side faces.

another area (not the apex of the droplet) is flattened by selforganization. When the droplet was placed on a tilted substrate, it became flat. The HFBI droplet was set on an FTO substrate at a tilt angle of 30°. From the apex of the droplet, a flat surface formed despite the tilted substrate (Figure 4). The flat face was 3701

DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704

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Figure 4. Behavior of an HFBI domical droplet on a tilted FTO substrate.

Figure 5. (a) Behavior of an HFBI domical droplet on an FTO substrate gently tilted from 0° to 25°. (b) Illustration of the behavior inside the HFBI droplet when the substrate was tilted.

Figure 6. Considerations of the behavior inside the HFBI droplet: (a) HFBI adsorbed at the air/water interface. (b) An HFBI molecule interacted with other surrounding HFBI molecules and was affected by buoyancy. (c) These HFBI units form precise membranes. The top of the domical droplet becomes flat.

densely packed membrane on the side of the HFBI droplet. If the side of the droplet had a dense HFBI membrane, the top self-organized HFBI membrane would not be able to move (the flat face would remain horizontal with respect to the substrate), whereas the flat face shifted to remain horizontal with the ground even when the substrate was tilted (Figure 5a). Hence, there was no densely packed membrane on the side of the HFBI droplet. This result corresponds to Figure 3. Behavior inside the HFBI Droplet. The behavior of HFBI in the water droplet was considered at the molecular level. First, HFBI adsorbed to the air/water interface (Figure 6a). Arrows mean a hydrophobic interaction. Next, the self-organized HFBI membrane assumed a very precise structure (Figure 1c) by which the intermolecular interaction of HFBI was considered to

horizontally oriented to the ground (perpendicular to the direction of gravitational force) but was not horizontal to the substrate. From this result, some force toward the apex is working in HFBI at an air/water interface. Next, a flat top HFBI droplet was gently tilted from 0° to 25°. The corresponding tilt time was 1 min. In this case, too, the flat face remained horizontal with respect to the ground even as the substrate was tilted (Figure 5a). This result suggests that the self-organized HFBI membrane was floating at the air/water interface similar to a raft (Figure 5b). In the case of HFBI, the working attraction (or driving) force directed toward the apex is considered to be buoyancy. In a previous study of the pendant drop method, the HFBI molecules were found to float upward to gather near the point of a needle.2,24,25 There was no 3702

DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704

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be very strong. Some adsorbed HFBI molecules at the air/water interface interacted with one another, and HFBI underwent a certain degree of accumulation at the interface. Buoyancy forces worked on this HFBI accumulation, and the gathering HFBI molecules floated to the top of the droplet (Figure 6b). The size of the HFBI molecule is about 2.3 nm × 2.3 nm × 2.67 nm (PDB data 2FZ6).7 The mass density of the HFBI molecule is 1.13 g/cm3, which is calculated from the molecular size and molecular mass. This value is close to the mass density of water (1 g/cm3). This fact means that the buoyancy of a single HFBI molecule is not enough to float in water. Single molecules of HFBI adsorb on air/water or water/solid interfaces by hydrophobic interaction (not by the buoyancy of HFBI). However, when HFBI molecules form a self-assembled membrane, buoyancy works at the air/water interface. Buoyancy can be formulated as

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F [N] = ρ [kg/m 3] × V [m 3] × g [m/s2]

The formula clearly shows that the self-assembled membrane form of HFBI had enough buoyancy in a water solvent. The molecules gathered at the apex were densely organized by the intermolecular interaction of HFBI. Finally, the top of the droplet was flattened due to the HFBI self-organization (Figure 6c). Arrows mean movement by buoyancy. The self-organized HFBI membrane at both the air/water interface and water/ solid interface is a monolayer.26−28

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CONCLUSION HFBI has a very unique feature, namely, the top of the domical droplet forms a flat surface. In this study, we clarified why only the top of the domical droplet was flattened but other areas of the droplet were not. The flat top region remained horizontal to the ground even when the substrate was tilted. Therefore, the force driving material up to the apex of the droplet was judged to be buoyancy. Initially, HFBI adsorbed on the air/ water interface; subsequently, each HFBI molecule interacted with nearby molecules, and finally, the HFBI units reached the apex of the droplet. To confirm the condition of the side face, a substrate that was transferred to this area was measured by AFM and EIS. From the results, it was confirmed that the top of the HFBI droplet had a densely packed, self-organized HFBI membrane, whereas the side of the HFBI droplet had no such membrane. Thus, the study results almost completely clarified the mechanism of the interface phenomenon for the HFBI domical droplet. This paper and our previous paper suggested that these studies were able to further the understanding of the behavior of molecules at the droplet interface, which becomes an indicator of special physical phenomena related to the droplet interface.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Japan Science and Technology Agency (JST) Advanced Catalytic Transformation Program for Carbon Utilization (ACT-C), the JST Japan-Finland Joint Research Program, and JSPS KAKENHI Grant Number 26600005. 3703

DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704

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DOI: 10.1021/acs.jpcb.6b01306 J. Phys. Chem. B 2016, 120, 3699−3704